This work was supported by the NSFC (No. U1532107) and Shanghai Jiao Tong University Biomedical Engineering project (No. YG2014MS53). The authors would like to acknowledge Jianying Li and Yan Shen for their helpful comments and technical support efforts in developing the DECT and PET/CT imaging method.

This work was performed in strict accordance with the standards established by the Guidelines for the Care and Use of Laboratory Animals of Shanghai Jiao Tong University and was approved by the laboratory Animal Ethics Committee of Ruijin Hospital.
1. Gastric Cancer Peritoneal Metastasis Animal Model
<ol>
	<li>Divide a moderately differentiated SGC-7901 human gastric cancer cell line into an SGC-7901/sFRP1 group and an SGC-7901/vector group. Culture the two groups of cells separately in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/mL streptomycin, and 100 &#181;g/mL penicillin at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
	<li>Use 4-6 week-old female athymic BALB/c nude mice with body weights of 25 to 30 g. Place animals under specific pathogen-free conditions in an animal facility.</li>
	<li>Divide mice randomly into the sFPR1 overexpression group and the sFPR1 empty loading group. 
	NOTE: Each group had ten nude mice; twenty mice were randomly divided into TGF-&#946;1 treatment group and TGF-&#946;1 control group, with up to 5 nude mice per animal cage.</li>
	<li>Establish the sFPR1-overexpression peritoneal metastasis xenograft models group by administering 150 &#181;L (2 x 106 cells/mL) suspensions of SGC-7901/sFRP1 cells via the abdominal cavity; SGC-7901/vector cells are administered to establish the empty loading group. 
	NOTE: Use a hemocytometer counting method to determine the concentration of cells20.</li>
	<li>Establish the peritoneal metastasis xenograft models group by administering 150 &#181;L (2 x 106 cells/mL) suspensions of SGC-7901 cells via the abdominal cavity. Administer the target therapy of TGF-&#946;1 inhibitor SB431542 after a period of two weeks of growth by intraperitoneal injection at a dose of 100 &#181;L/10 g of body weight every other day to mice in the treatment group.
	<ol>
		<li>Administer normal saline at the same dose to mice in the control group.</li>
	</ol>
	</li>
	<li>Perform the DECT and PET/CT scanning 1 day before treatment and 1 day, 7 days, 14 days, and 21 days after treatment.</li>
</ol>
2. DECT for Peritoneal Metastasis Animal Model
NOTE: The animal imaging experiment was achieved on the dual energy CT scanner (see Table of Materials). We created the related DECT imaging protocol according to the previous studies.
<ol>
	<li>Setup for DECT imaging protocol
	<ol>
		<li>On the imaging console computer, select the &#34;protocol management&#34; icon to enter the next interface, then click the &#34;protocol management&#34; option to view the protocol management screen.</li>
		<li>In the &#39;User protocol&#39; interface, select the abdomen area to enter the abdomen protocols list.</li>
		<li>Click the blank space in the protocol lists and select the &#34;New&#34; button to type the name of a new protocol: &#34;Animal DECT Scan&#34;. Press the &#34;Enter&#34; key on the keyboard and select the &#34;Scout&#34; button in the pop-up window, click &#34;OK&#34; to setup the scout series (the first series).</li>
		<li>Select the &#34;XY&#34; mode for &#39;Anatomical Reference Point&#39; and the &#34;head-first supine position&#34; for &#39;Patient Orientation&#39;. Click on the &#34;auto transfer&#34; option and select the workstation location where the image series will be uploaded. Name &#34;Scout phase&#34; in Series Description.</li>
		<li>In the &#39;View Edit&#39; screen, make sure the relevant scan parameters are set as the following: &#34;Start location&#34; and &#34;End Location&#34; options are set at &#34;S50&#34; and &#34;I50&#34; respectively, &#34;KV&#34; at &#34;100&#34;, &#34;mA&#34; at &#34;80&#34;, &#34;90&#176;&#34; for &#34;Lateral scout position&#34;, &#34;0&#176;&#34; for &#34;AP scout position&#34;, and the &#34;scout WW/WL&#34; at &#34;400/40&#34;.</li>
		<li>Next, create the second series for non-enhanced scanning. Click &#34;Create New Series&#34;, in the pop-up window to select the &#34;Axial&#34; and &#34;Create After&#34; icons.</li>
		<li>Name the series as &#34;-C phase&#34; in the Series Description and turn &#34;Show localizer&#34; on. In the Scan type interface, select the &#34;helical&#34; scan type and 0.5 s for &#34;Rotation Time&#34;, click &#34;Thick Speed&#34; option to set the parameters (Detector Coverage at 40 mm, Helical Thickness at 0.625 mm, Pitch and speed at 0.516:1/20.62, Rotation Time at 0.5 s) in the pop-up window, Interval at 0.625 mm, Gantry Tit at 0, SFOV select Small Body, kV at 100, click &#34;mA&#34; then type 600 for Manual mA.</li>
		<li>Click the &#34;Recon Parameters&#34; icon and open the &#34;Recon Option&#34; pop-up window. Select &#34;Plus&#34; in Recon Mode; click the &#34;Slice&#34; icon to select &#34;ss50 slice 50%&#34; mode in the &#39;ASiR Setup screen&#39;. Set the remaining parameters as follows: DFOV at 25 cm, R/L and A/P Center at 0 cm, Recon type select &#34;Stnd&#34;, Matrix Size at 512.</li>
		<li>Create the third scan series for enhanced scanning by repeating the step 2.1.8, name it as &#34;+C QC phase&#34; and turn &#34;Show localizer&#34; on; the first group is the setup of arterial phase series.</li>
		<li>In the &#39;Scan type&#39; interface, click on &#34;GSI (Gemstone Spectral Imaging)&#34; and select &#34;helical&#34; scan type, select the protocol &#34;GSI-52&#34; in the &#39;Abdomen GSI Preset Selection&#39; window. Set start Location and end Location according to the non-enhanced scanning.
		<ol>
			<li>Click the &#34;Recon Parameters&#34; icon and open the &#34;Recon Option&#34; pop-up window. In Recon Mode, select &#34;Plus&#34; and in &#39;GSI option&#39; click &#34;QC&#34;; the remaining parameters are the same as in step 2.1.8.</li>
		</ol>
		</li>
		<li>Click the &#34;R2&#34; icon and select &#34;YES&#34; in the &#34;Recon enabled&#34; tab. Select &#34;Thickness&#34; at 0.625 and type 0.625 for &#34;Interval&#34;. Open the &#34;Recon Option&#34; pop-up window, in Recon Mode select &#34;Plus&#34;; in GSI options, click &#34;Mono&#34; and set the keV to 70 keV; in the ASiR Setup window, select the &#34;GS40 40%&#34; mode for the GSI ASiR setup. The remaining parameters are set with step 2.1.8. Name this step as &#34;+C 70keV phase&#34;.</li>
		<li>Click the &#34;R3&#34; icon and select &#34;YES&#34; in the &#34;Recon enabled&#34; tab. Set &#34;Thickness&#34; at 1.25 and type 0.625 for &#34;Interval&#34;. Open the &#34;Recon Option&#34; pop-up window, in Recon Mode select &#34;Plus&#34; and &#34;IQ enhanced&#34;; in the GSI options, click &#34;Mono&#34;, set the keV to 70 keV and click &#34;GSI Data File&#34;; ensure the GSI ASiR setup is in accordance with step 13. The remaining parameters are set as in step 2.1.8. Name this as &#34;+C Mono phase&#34;.</li>
		<li>Click &#34;Add group&#34; to create two scan groups to represent the portal phase and the delay phase, respectively. Make sure that the &#34;Start Location&#34; and &#34;End Location&#34; ranges of each scan phase are consistent and the remaining parameters are the same as the arterial phase. Type the delay time in &#34;Preparation Group&#34;: the first group (arterial phase) at 0 s, the second group (portal phase) at 8 s, and the third group (delay phase) at 16 s.</li>
		<li>Click the &#34;Accept&#34; option to save the protocol after all the settings are done.</li>
	</ol>
	</li>
	<li>DECT imaging process
	<ol>
		<li>Select the nude mice randomly from the treatment and control groups before each scan. Place the selected animals in new cages and mark them separately.</li>
		<li>Fast the mice for 4 h with water but without food or bedding.</li>
		<li>Remove the experimental mice from the animal experiment center 1 h before the scan, and make sure the mice are placed in a new warm environment until the scan begins.</li>
		<li>Anesthetize all the experimental mice with an intraperitoneal injection of 2.5% pentobarbital sodium (1.0 mL/kg body weight) before DECT scan imaging, and confirm the depth of anesthesia by the toe pinch reflex. Use ointment on the eyes to prevent dryness while under anesthesia. 
		NOTE: Ensure that the head of each nude mouse is in the lower position when injecting the drugs, thus reducing damage to the internal organs. Pay attention to the injection site and the depth of injection. Place the tip of the syringe at a 45&#176; angle to the inside of the right/left lower abdomen, and ensure that the needle depth is such that injection into the bowel and other organs is avoided.</li>
		<li>Click the &#34;New Patient&#34; icon, input the basic information about the mouse including its patient ID and name. In the &#39;User protocol&#39;, click the &#34;abdomen protocol&#34; and select the &#34;Animal DECT Scan&#34; protocol to enter the operation interface.</li>
		<li>Once anesthesia is induced, move each mouse onto an animal fixture platform in the supine position, and fix its tail with tape to make sure it does not bend. Sterilize the tail with alcohol for subsequent contrast agent injection into the tail-vein.</li>
		<li>Move the CT scan bed so that the external positioning line laser is over the lower abdomen of the animal. Click the &#34;reset&#34; button when the positioning is completed. 
		NOTE: The placement of external positioning lines over the lower abdomen of the animal ensures that the animals are located as far as possible on the outside of the machine for easy contrast agent administration into the tail vein.</li>
		<li>Click the &#34;Confirm&#34; icon and follow the flashing order of the buttons on the keyboard to complete the scout scanning. Select the &#34;next series&#34; icon after the scout scanning is completed, and enter the interface for non-enhanced scanning.</li>
		<li>In the right screen, set the &#34;Start Location&#34; and &#34;End Location&#34; on the scout views to define the scan range. Maintain the same range in &#39;Lateral scout&#39; and &#39;AP scout&#39; and cover the entire body volume of the animal.</li>
		<li>Click the &#34;Confirm&#34; icon and follow the flashing order of the buttons on the keyboard to complete the scout scanning.</li>
		<li>Inject each mouse with iopamidol at a dosage of 0.2 mL/100 g through the tail vein. 
		NOTE: We chose to administer the contrast medium manually and keep the injection rate as stable as possible. It is most conducive to capture the early enhancement of the tumor during imaging.</li>
		<li>Click &#34;next series&#34; to perform enhanced scanning. Set &#34;Start Location&#34; and &#34;End Location&#34; as per the non-enhanced scan. Click the &#34;confirm&#34; icon and follow the flashing order of the buttons on the keyboard to complete the dynamic enhanced scans, includingarterial phase, portal phase, and delayed phase. 
		NOTE: Click the &#34;Confirm&#34; icon immediately to start scanning after the contrast agent is injected. This is essential and crucial for enhanced scanning to ensure the best image of the arterial phase is captured. However, some delay time after clicking the &#34;confirm&#34; icon can ensure that the experimental staff has withdrawn safely from the scanning room.</li>
		<li>Click &#34;End exam&#34; to exit the scanning interface after the scanning is complete; the image series will be automatically uploaded to the workstation.</li>
		<li>Place the animal into an empty cage after completion of the scan with all mice, and observe them until they have regained consciousness.Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Then transfer the mice into a clean animal room.</li>
	</ol>
	</li>
	<li>Post DECT imaging analysis
	<ol>
		<li>Locate the mice series on the DECT workstationinterface (see Table of Materials) and select the &#34;+C Mono phase&#34; series lists. Open &#34;GSI Volume Viewer&#34; and select &#34;GSI VV General&#34; protocol from the &#39;GSI Protocol Manager&#39; interface.</li>
		<li>Click the &#34;View Type&#34; active annotation at the upper-left corner of the image viewports and select &#34;coronal&#34; orientation from the drop-down menu.</li>
		<li>For one image viewport, click the &#34;Volume 1&#34; active annotation at the upper-left corner and select &#34;Mono&#34; volumes from the drop-down menu. Similarly, in another image viewport, select &#34;Iodine (Water)&#34; volumes. Click and hold the left mouse button, drag the image from &#34;Iodine (Water)&#34; viewport to &#34;Mono&#34; and check the &#34;mix the views&#34; box to get the color fused images.</li>
		<li>Click and drag from the center of the &#34;Image Scroll&#34; icon to observe images. Save the images which show positive results as color fused images.</li>
	</ol>
	</li>
</ol>
3. PET/CT for Peritoneal Metastasis Animal Model
NOTE: See the table of materials for the PET/CT imager used. We created the related PET/CT imaging protocol according to this article21.
<ol>
	<li>Setup Micro-PET/CT Imaging Protocol
	<ol>
		<li>For a whole-body CT scan, set current at 500 &#181;A, voltage at 80 kV, exposure time at 200 ms, and 240 steps for 240&#176; rotation. For X-ray detector, select resolution at &#34;low system magnification&#34; with 78 mm axial imaging field and single bed mode. Use the &#34;Common Cone-Beam Reconstruction&#34; method and select the &#34;real time reconstruction&#34; option, so that the host PC can connect with the dedicated real time reconstruction computer (Cobra) to initiate the task.</li>
		<li>For PET acquisition, in the &#34;acquire by time&#34; option set &#34;fixed scan time&#34; to 600 s (10 min). Set &#34;study isotope&#34; to F-18 and &#34;energy level&#34; to 350-650 keV.</li>
		<li>To produce the PET Histogram, set the &#34;dynamic frame&#34; as &#34;black&#34; to process data as one frame for the entire duration to achieve static scan. Set histogram type to &#34;3D&#34; and select the &#34;no scatter correction&#34; option.</li>
		<li>For PET reconstruction, reconstruct images using an OSEM3D algorithm followed by MAP or Fast MAP22 provided by PET/CT workstationsoftware (see Table of Materials).</li>
	</ol>
	</li>
	<li>Preparation before PET/CT imaging
	<ol>
		<li>Fast the mice which have undergone DECT experiments for 4 h and transfer the mice to new animal cages 30 min before imaging.</li>
		<li>Weigh the mice and record their weight.</li>
		<li>Follow the institute&#39;s safety procedures to acquire and carry the package containing radioactive materials (RAM). Use a protective shield to carry the 18F-FDG (5 mCi), and measure the radioactivity of the total 18F-FDG with a dose calibrator.</li>
		<li>Dilute the 18F-FDG with normal saline to the appropriate radioactivity of mice injection. 
		NOTE: The diluted activity of 18F-FDG should be available at 100-200 &#181;Ci/100 &#181;L for each mouse.
		<ol>
			<li>Draw 200 &#181;L 18F-FDG solution into a 1 mL syringe. Measure the radioactivity of the whole syringe with a dose calibrator and record the 18F-FDG preparation time.</li>
		</ol>
		</li>
		<li>Inject each mouse with 200 &#181;L 18F-FDG solution via the tail intravenous injection route and record the 18F-FDG injection time. After the injection of all mice, measure the residual radioactivity of the syringe with the dose calibrator immediately and record the time that the measurements were taken after completion of the injection.</li>
		<li>Calculate the injected 18F-FDG activity for each mouse by the following formula: Injected activity (&#181;Ci) = activity in syringe before injection - activity in syringe after injection.</li>
	</ol>
	</li>
	<li>PET/CT Imaging Process
	<ol>
		<li>Put the animal into an anesthesia induction chamber; anesthetize mouse using inhaled 3% Isoflurane in oxygen after the completion of 18F-FDG injection. 
		NOTE: Follow all animal welfare guidelines appropriate for operation; keep the mice warm by using the heating pad. Use ointment on eyes to prevent dryness while under anesthesia.</li>
		<li>Once anesthesia is induced, move the mouse onto the micro-CT scanning bed while maintaining continuous anesthesia and warming. Position the head of the mouse within a cone face mask that continuously delivers Isoflurane (2%) in oxygen at a flow rate of 2 L/min. Place the mouse in supine position to ensure the posture is consistent with that in DECT scans.</li>
		<li>Move the animal to the entrance of the PET/CT scanner, click the &#34;laser&#34; icon from the toolbar viewer, and use the touchpad control interface to move the bed so that the abdomen of the mouse is located at the center of the PET and CT field-of-view (FOV) during scanning. In the &#34;Laser Align&#34; window, select &#34;first scan type&#34; as &#34;CT scan&#34;, and &#34;PET acquisition included in workflow&#34; as the option.</li>
		<li>Open the &#34;Scout View&#34; window and acquire a scout view X-ray radiograph. Adjust the position of the animal bed so that the center field of view of the CT is located at the center of the mouse body.</li>
		<li>Select the protocol established previously (in step 3.1). Input the number of mice to be imaged (successively) in the pop-up window and click the &#34;Setup&#34; option, then enter the weight. Then click the &#34;Setup&#34; option again and follow the pop-up window instructions to complete the setup.</li>
		<li>Click the &#34;Start Workflow&#34; icon to start scanning.</li>
		<li>Evaluate the quality of the acquired CT and PET images after all the scans are completed. Transfer the data through the network to the post imaging analysis for further study. 
		NOTE: Adjust the window width and window level of the image to ensure that the contrast of the organs is displayed properly. Check the resolution of the organs in the images to confirm the imaging quality.</li>
		<li>Remove the animal from the imager and immediately euthanize by cervical dislocation. Use the imaging system for the next animal successively.</li>
	</ol>
	</li>
	<li>Post PET/CT imaging analysis
	<ol>
		<li>Open the PET/CT workstation software, import the CT and PET image series data into the software. In the &#34;Registration&#34; window, click the &#34;General analysis&#34; option to register CT and PET images together, and choose the &#34;Sky&#34; model under the &#34;Review&#34; window to show a perfect alignment between CT and PET images.</li>
		<li>Identify the peritoneal nodules with references provided by the co-registered images in the &#34;Region of Interest (ROI) Quantification&#34; window.</li>
		<li>In the &#34;Region of Interest (ROI) Quantification&#34; window, draw ROI with tools over the fused images, edit the size and shape of ROI, record the SUVmax value, then output and save the selected merged images.</li>
	</ol>
	</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+functional+imaging+in+the+management+of+lymphoma.">Role of functional imaging in the management of lymphoma.</a> J Clin Oncol. 29 (14), 1844-1854 (2011).</li><li>Fuster, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preoperative+staging+of+large+primary+breast+cancer+with+[18F]fluorodeoxyglucose+positron+emission+tomography/computed+tomography+compared+with+conventional+imaging+procedures.">Preoperative staging of large primary breast cancer with [18F]fluorodeoxyglucose positron emission tomography/computed tomography compared with conventional imaging procedures.</a> J Clin Oncol. 26 (29), 4746-4751 (2008).</li><li>Lin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+frizzled-related+protein+1+overexpression+in+gastric+cancer:+Relationship+with+radiological+findings+of+dual-energy+spectral+CT+and+PET-CT.">Secreted frizzled-related protein 1 overexpression in gastric cancer: Relationship with radiological findings of dual-energy spectral CT and PET-CT.</a> Scientific Reports. 7, 42020 (2017).</li><li>Cadena-Herrera, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+three+viable-cell+counting+methods:+Manual,+semi-automated,+andautomated.">Validation of three viable-cell counting methods: Manual, semi-automated, andautomated.</a> Biotechnol Rep (Amst). 7, 9-16 (2015).</li><li>Wang, X., Minze, L. J., Shi, Z. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+brown+fat+in+mice+with+18F-FDG+micro-PET/CT.">Functional imaging of brown fat in mice with 18F-FDG micro-PET/CT.</a> J Vis Exp. (69), (2012).</li><li>Grootjans, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+3DOSEM+and+MAP+algorithms+for+reconstructing+low+count+SPECT+acquisitions.">Performance of 3DOSEM and MAP algorithms for reconstructing low count SPECT acquisitions.</a> Z Med Phys. 26 (4), 311-322 (2016).</li><li>Chang, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+gastric+cancer+preclinical+studies+using+diverse+in+vitro+and+in+vivo+model+systems.">Improving gastric cancer preclinical studies using diverse in vitro and in vivo model systems.</a> BMC Cancer. 16, 200 (2016).</li><li>Chang, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HNF4alpha+is+a+therapeutic+target+that+links+AMPK+to+WNT+signalling+in+early-stage+gastric+cancer.">HNF4alpha is a therapeutic target that links AMPK to WNT signalling in early-stage gastric cancer.</a> Gut. 65 (1), 19-32 (2016).</li><li>Zheng, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BTG1+expression+correlates+with+pathogenesis,+aggressive+behaviors+and+prognosis+of+gastric+cancer:+a+potential+target+for+gene+therapy.">BTG1 expression correlates with pathogenesis, aggressive behaviors and prognosis of gastric cancer: a potential target for gene therapy.</a> Oncotarget. 6 (23), 19685-19705 (2015).</li><li>Yamada, A., Oguchi, K., Fukushima, M., Imai, Y., Kadoya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+2-deoxy-2-[18F]fluoro-D-glucose+positron+emission+tomography+in+gastric+carcinoma:+relation+to+histological+subtypes+depth+of+tumor+invasion,+and+glucose+transporter-1+expression.">Evaluation of 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography in gastric carcinoma: relation to histological subtypes depth of tumor invasion, and glucose transporter-1 expression.</a> Ann Nucl Med. 20 (9), 597-604 (2006).</li><li>Hirose, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+2-deoxy-2-[(18)F]-fluoro-d-glucose+uptake+and+clinicopathological+factors+in+patients+with+diffuse+large+B-cell+lymphoma.">Relationship between 2-deoxy-2-[(18)F]-fluoro-d-glucose uptake and clinicopathological factors in patients with diffuse large B-cell lymphoma.</a> Leuk Lymphoma. 55 (3), 520-525 (2014).</li><li>Tchou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degree+of+tumor+FDG+uptake+correlates+with+proliferation+index+in+triple+negative+breast+cancer.">Degree of tumor FDG uptake correlates with proliferation index in triple negative breast cancer.</a> Mol Imaging Biol. 12 (6), 657-662 (2010).</li><li>Coleman, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+PET/CT+with+an+integrated+imaging+system:+intersociety+dialogue+from+the+Joint+Working+Group+of+the+American+College+of+Radiology+the+Society+of+Nuclear+Medicine,+and+the+Society+of+Computed+Body+Tomography+and+Magnetic+Resonance.">Concurrent PET/CT with an integrated imaging system: intersociety dialogue from the Joint Working Group of the American College of Radiology the Society of Nuclear Medicine, and the Society of Computed Body Tomography and Magnetic Resonance.</a> J Am Coll Radiol. 2 (7), 568-584 (2005).</li><li>Brepoels, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+corticosteroids+on+18F-FDG+uptake+in+tumor+lesions+after+chemotherapy.">Effect of corticosteroids on 18F-FDG uptake in tumor lesions after chemotherapy.</a> J Nucl Med. 48 (3), 390-397 (2007).</li><li>Spaepen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[18)F]FDG+PET+monitoring+of+tumour+response+to+chemotherapy:+does+[(18)F]FDG+uptake+correlate+with+the+viable+tumour+cell+fraction.">[18)F]FDG PET monitoring of tumour response to chemotherapy: does [(18)F]FDG uptake correlate with the viable tumour cell fraction.</a> Eur J Nucl Med Mol Imaging. 30 (5), 682-688 (2003).</li></ol>

The authors have no conflicts of interest to declare.

Animal models have been used widely in the study of molecular mechanisms underlying gastric cancer, and to experiment with various therapeutic strategies23,24,25. In this study, we have described a detailed protocol for gastric cancer peritoneal metastasis nude mice modeling, using DECT and PET/CT to image gastric tumors for identifying tumor cell proliferation in real-time, and monitoring peritoneal metastasis and responses to ...

Gastric cancer (GC) remains the fourth most common malignancy and the second-leading cause of cancer mortality worldwide1. Although the accuracy in diagnosis and treatment of gastric cancer has been greatly improved, peritoneal metastasis is the most key point of gastric cancer prognosis or recurrence and is a definitive determinant of postoperative death2. It is generally accepted that peritoneal dissemination is a life-threatening mode of metastasis, wherein the disease becomes uncontrollable and the prognosis of the patient is poor once peritoneal dissemination is established. Therefore, the detection and therapeutic effect evaluation of gastric cancer peritoneal metastasis is crucial for clinical practice.
The increasing incidence and mortality of gastric cancer had spurred researchers to identify its molecular mechanisms. The high expression of genes such as secreted frizzled-related protein 1 (sFRP1) might lead to activation of the signal pathway in the early stages of gastric cancer, promoting the process of tumor growth, proliferation, differentiation, and apoptosis3,4,5,6,7. sFRP1-overexpression cells showed an increase in the expression of TGF&#946;, its downstream targets, and TGF&#946;-mediated EMT8. Previous studies have demonstrated that the TGF-&#946;1 level is correlated with peritoneal metastasis and the TNM stages of gastric cancer. We have described the changes in cancer cell proliferation regulated by sFRP1 overexpression and TGF-&#946;1 inhibition, and established animal models for peritoneal metastasis to show the performance of tumor imaging under the effects of gene regulation.
Animal models for gastric cancer are indispensable tools for researching tumor development and experimenting with various therapeutic strategies without having to sacrifice animals. Animal models have proven useful in studying the formation mechanisms of tumors and cells of origin, determining the presence of cancer stem cells, and examining various novel therapeutic strategies. Therefore, a real-time non-invasive technique can provide an accurate description of the development of gastric tumors and tumor response to treatments, which can identify the development of peritoneal metastasis nodules in nude mice and monitor the changes of a tumor in response to various experimental and therapeutic interventions.
Currently, multi-detector CT (MDCT) plays an important role in the TNM staging of gastric cancers and is useful for predicting tumor resectability preoperatively9. However, radiological studies of patients with histologically proven gastric carcinoma have mainly been based on morphology. DECT imaging extends the parameters to reflect functional information by providing monochromatic images and may be helpful for improving the N staging accuracy for gastric cancers. Furthermore, this technique will enable the acquisition of material-decomposition images, which may be useful to differentiate between differentiated and undifferentiated gastric carcinoma, and between metastatic and non-metastatic lymph nodes10. With the introduction of DECT, the functional imaging aspect of CT has also been added to clinical applications, contributing to evaluations of therapeutic efficacy and predicting patient prognoses11,12,13. PET/CT is a useful imaging technique for the detection and staging of gastric cancer and can evaluate the recurrence of the tumor effectively14. Tumor cell proliferation and angiogenesis were both considered to be necessary in the development of a detectable tumor15, tumor nodules showed a positive performance with higher SUVmax on PET/CT. Based on their preference for aerobic glycolysis, 18F-FDG, a glucose analog, has been exploited as a promising tracer in the diagnosis of malignancies, combined with PET/CT16. This method relies on the rapid glucose consumption of tumor tissue and has broad clinical applications, including assisting in the detection, staging, and evaluation of the prognosis of tumors, as well as monitoring the tumors&#39; response to therapy17,18. As non-invasive methods, DECT and PET/CT have been utilized to diagnose malignant tumors and to assess tumor response to various therapies.
Our group has been using this non-invasive imaging method with DECT and PET/CT scanners to detect and monitor the process of tumor growth and metastasis in living mice19. We explored imaging findings induced by the sFRP1-overexpression in gastric cancer cells in vivo using nude mice, with DECT and PET/CT, and described the changes of the SUVmax value following targeted therapy by the TGF-&#946;1 inhibitor to confirm the development of tumor nodules in the peritoneum after gene induction, and also studied the changes in tumor nodules in response to experimental treatments. In this paper, we present detailed procedures for modeling gastric tumor peritoneal metastasis in mice, and its detection and monitoring with DECT and PET/CT.

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			<td>dual-energy spectral CT workstation</td>
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			<td height="21" style="height:21px;width:247px;">Siemens Inveon micro-PET/CT</td>
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			<td>positron emission tomography/ 
			computed tomography scanner&#160;</td>
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			<td style="width:389px;">Siemens Preclinical Solution</td>
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gene regulation and targeted therapy in gastric cancer peritoneal metastasis: radiological findings from dual energy ct and pet/ct

Gastric cancer remains fourth in cancer incidence worldwide with a five-year survival of only 20%-30%. Peritoneal metastasis is the most frequent type of metastasis that accompanies unresectable gastric cancer and is a definitive determinant of prognosis. Preventing and controlling the development of peritoneal metastasis could play a role in helping to prolong the survival of gastric cancer patients. A non-invasive and efficient imaging technique will help us to identify the invasion and metastasis process of peritoneal metastasis and to monitor the changes in tumor nodules in response to treatments. This will enable us to obtain an accurate description of the development process and molecular mechanisms of gastric cancer. We have recently described experiment using dual energy CT (DECT) and positron emission tomography/computed tomography (PET/CT) platforms for the detection and monitoring of gastric tumor metastasis in nude mice models. We have shown that weekly continuous monitoring with DECT and PET/CT can identify dynamic changes in peritoneal metastasis. The sFRP1-overexpression in gastric cancer mice models showed positive radiological performance, a higher FDG uptake and increasing enhancement, and the SUVmax (standardized uptake value) of nodules demonstrated an obvious alteration trend in response to targeted therapy of TGF-&#946;1 inhibitor. In this article, we described the detailed non-invasive imaging procedures to conduct more complex research on gastric cancer peritoneal metastasis using animal models and provided representative imaging results. The use of non-invasive imaging techniques should enable us to better understand the mechanisms of tumorigenesis, monitor tumor growth, and evaluate the effect of therapeutic interventions for gastric cancer.

DECT and PET/CT scanning were performed on nude mice after two weeks of cell line injections. GSI images yielded excellent results for displaying subcutaneous metastasis beyond the contour of the abdomen for the sFRP1 overexpression group, and metastasis with peripheral enhancement was confirmed by color-scale image (Figure 1a-c). PET/CT images depicted focally abnormal FDG uptake of metastasis, including in the peritoneal and subcutaneous me...

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Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT

This protocol describes the value of dual energy CT and PET/CT imaging methods in tumor imaging and efficacy evaluation. This article demonstrates the research methods and results acquired by dual energy CT and PET/CT to evaluate the gene regulation and targeted treatment of gastric cancer peritoneal metastasis.

Gastric cancer remains fourth in cancer incidence worldwide with a five-year survival of only 20%-30%. Peritoneal metastasis is the most frequent type of ...

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10.7K Views.  Shanghai Jiao Tong University School of Medicine. This protocol describes the value of dual energy CT and PET/CT imaging methods in tumor imaging and efficacy evaluation. This article demonstrates the research methods and results acquired by dual energy CT and PET/CT to evaluate the gene regulation and targeted treatment of gastric cancer peritoneal metastasis.

Video: Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT)

Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1 and carotid intimal medial thickness (C-IMT)2 provide information about the burden of disease. However, despite multiple validation studies of CAC3-5, and C-IMT2,6, these modalities do not accurately assess plaque characteristics7,8, and the composition and inflammatory state of the plaque determine its stability and, therefore, the risk of clinical events9-13.
[18F]-2-fluoro-2-deoxy-D-glucose (FDG) imaging using positron-emission tomography (PET)/computed tomography (CT) has been extensively studied in oncologic metabolism14,15. Studies using animal models and immunohistochemistry in humans show that FDG-PET/CT is exquisitely sensitive for detecting macrophage activity16, an important source of cellular inflammation in vessel walls. More recently, we17,18 and others have shown that FDG-PET/CT enables highly precise, novel measurements of inflammatory activity of activity of atherosclerotic plaques in large and medium-sized arteries9,16,19,20. FDG-PET/CT studies have many advantages over other imaging modalities: 1) high contrast resolution; 2) quantification of plaque volume and metabolic activity allowing for multi-modal atherosclerotic plaque quantification; 3) dynamic, real-time, in vivo imaging; 4) minimal operator dependence. Finally, vascular inflammation detected by FDG-PET/CT has been shown to predict cardiovascular (CV) events independent of traditional risk factors21,22 and is also highly associated with overall burden of atherosclerosis23. Plaque activity by FDG-PET/CT is modulated by known beneficial CV interventions such as short term (12 week) statin therapy24 as well as longer term therapeutic lifestyle changes (16 months)25.
The current methodology for quantification of FDG uptake in atherosclerotic plaque involves measurement of the standardized uptake value (SUV) of an artery of interest and of the venous blood pool in order to calculate a target to background ratio (TBR), which is calculated by dividing the arterial SUV by the venous blood pool SUV. This method has shown to represent a stable, reproducible phenotype over time, has a high sensitivity for detection of vascular inflammation, and also has high inter-and intra-reader reliability26. Here we present our methodology for patient preparation, image acquisition, and quantification of atherosclerotic plaque activity and vascular inflammation using SUV, TBR, and a global parameter called the metabolic volumetric product (MVP). These approaches may be applied to assess vascular inflammation in various study samples of interest in a consistent fashion as we have shown in several prior publications.9,20,27,28

Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography FDG-PET/CT

There is great need to identify atherosclerosis non-invasively, and here we demonstrate how FDG-PET/CT can be used to detect and quantify atherosclerotic plaque activity and vascular inflammation.

Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1 and carotid intimal medial thickness (C-IMT)2 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...